PROJECT SUMMARY The overall goal of this project is to engage undergraduate and graduate students in innovative studies in molecular toxinology, studies that have the potential to contribute to important advances in our understanding of the pathophysiology of snakebite. Snakebite is a substantial global health problem, causing severe injury to 2.7 million people and claiming an estimated 125,000 deaths annually. In spite of its importance as a global health issue, very little is known on the pathophysiology of snakebite. The goal of this project is to understand the effects of snake venom Cysteine-Rich Secretory Proteins (svCRiSPs) on vascular biology and the possible role that these effects may play in the pathophysiology of snakebite. The data generated from our previous award (R15HL137134-01) has demonstrated that svCRiSP toxins have an acute effect on vascular permeability. In the forthcoming grant period, we will build upon our previous studies with student-led projects to characterize the comparative vascular activity of multiple svCRiSPs isolated from the venoms of North American snakes housed in the NNTRC. In addition we will pursue our recent discovery that extracellular vesicles (EVs) isolated from the plasma of envenomated mice contain snake venom proteins including, prominently svCRiSPs. This new finding raises the possibility that venom proteins are transported in the systemic circulation by membrane vesicle trafficking. svCRiSP EVs may play in important role in regulating immune system activity impacting vascular permeability, pro-inflammatory responses, and the progression of physiological effects in snakebite. To test our hypothesis we will address three distinct but complementary Specific Aims: Specific Aim 1: To determine the role of exosomal CRiSPs in the pathophysiology of snakebite by comparing the distribution of exosomal CRiSPs in crotaline snake envenomation and evaluate the functional vascular effect of EVs from mice treated with svCRiSPs. Specific Aim 2: to characterize the effects of Css-CRiSP on the induction of pro-inflammatory responses in vitro and in vivo. Specific Aim 3: to profile the vascular effects, molecular mechanisms, and targets of crotaline svCRiSPs prepared from additional medically significant species of North American vipers. The proposed studies will support the educational goals of our program by providing undergraduate and graduate students with hands-on research experiences that will provide an introduction to the opportunities available for careers in biomedical research. This research program will also enhance the academic environment of our university by promoting an important program of global health research. Knowledge gained from these studies will generate a more detailed understanding of the effects of svCRiSPs on vascular biology and provide a better understanding of the role svCRiSPs play in the pathophysiology of snakebite.